Human ES (hES) cells have been shown to generate the entire range of major somatic cell lineages of the CNS (Reubinoff et al., “Embryonic Stem Cell Lines from Human Blastocysts: Somatic Differentiation In Vitro,” Nat Biotechnol 18:399-404 (2000); Zhang et al., “In vitro Differentiation of Transplantable Neural Precursors from Human Embryonic Stem Cells,” Nat Biotechnol 19:1129-1133 (2001); Schuldiner et al., “Induced Neuronal Differentiation of Human Embryonic Stem Cells,” Brain Res 913:201-205 (2001); Carpenter et al., Characterization and Differentiation of Human Embryonic Stem Cells,” Cloning Stem Cells 5:79-88 (2003); Park et al., “Generation of Dopaminergic Neurons In Vitro from Human Embryonic Stem Cells Treated with Neurotrophic Factors,” Neurosci Lett 359:99-103 (2004)) and thus represent a potentially important source for cell-based therapies of CNS diseases. Most of the studies aimed at generating CNS specific phenotypes from hES cells have succeeded in partially directing these cells towards generation of neural stem cells (NSC), which have then been shown to give rise to a mixture of neural phenotypes. However, the selective production and isolation of specific clinically important neuronal and glial phenotypes from hES cells is yet to be accomplished. Indeed, to date no prospectively defined neuronal phenotype has yet been either induced or selected from hES cell cultures.
In contrast, a variety of specific neuronal phenotypes have been selectively induced in murine ES cell cultures using extracellular factors (Brustle et al., “Embryonic Stem Cell-derived Glial Precursors: A Source of Myelinating Transplants,” Science 285:754-756 (1999); Kawasaki et al., “Induction of Midbrain Dopaminergic Neurons from ES Cells by Stromal Cell-derived Inducing Activity,” Neuron 28:31-40 (2000); Lee et al., “Efficient Generation of Midbrain and Hindbrain Neurons from Mouse Embryonic Stem Cells,” Nat Biotechnol 18:675-679 (2000); Liu et al., “Embryonic Stem Cells Differentiate into Oligodendrocytes and Myelinate in Culture and After Spinal Cord Transplantation,” Proc Natl Acad Sci USA 97:6126-6131 (2000); Wichterle et al., “Directed Differentiation of Embryonic Stem Cells into Motor Neurons,” Cell 110:385-397 (2002); Barberi et al., “Neural Subtype Specification of Fertilization and Nuclear Transfer Embryonic Stem Cells and Application in Parkinsonian Mice,” Nat Biotechnol 21:1200-1207 (2003), or a combination of extracellular factors and genetic manipulation (Kim et al., “Dopamine Neurons Derived from Embryonic Stem Cells Function in an Animal Model of Parkinson's Disease,” Nature 418:50-56 (2002); Billon et al., “Normal Timing of Oligodendrocyte Development from Genetically Engineered, Lineage-selectable Mouse ES Cells,” J Cell Sci 115:3657-3665 (2002). In particular, both dopaminergic (Lee et al., “Efficient Generation of Midbrain and Hindbrain Neurons from Mouse Embryonic Stem Cells,” Nat Biotechnol 18:675-679 (2000)) and cholinergic motor neurons (Wichterle et al., “Directed Differentiation of Embryonic Stem Cells into Motor Neurons,” Cell 110:385-397 (2002)), expressing transcription factors typical of midbrain and spinal cord cells, respectively, have been selectively induced. In both cases, using key stage-specific factors well described in developmental mouse models (Durston et al., “Retinoic Acid Causes an Anteroposterior Transformation in the Developing Central Nervous System,” Nature 340:140-144 (1989); Ericson et al., “Pax6 Controls Progenitor Cell Identity and Neuronal Fate in Response to Graded Shh Signaling,” Cell 90:169-180 (1997); Ye et al., “FGF and Shh Signals Control Dopaminergic and Serotonergic Cell Fate in the Anterior Neural Plate,” Cell 93:755-766 (1998); Muhr et al., “Assignment of Early Caudal Identity to Neural Plate Cells by a Signal from Caudal Paraxial Mesoderm,” Neuron 19:487-502 (1999)), the two ventral neuronal phenotypes were generated: in the former case, dopaminergic neurons expressing transcription factors typical of the ventral mesencephalon were induced under the combined influence of sonic hedgehog (SHH) and FGF8, while in the latter, spinal cord motor neurons were generated by a combined treatment of SHH and RA. These studies show that signaling pathways delineated from various in vivo models of development can be used, to a certain limit, to selectively drive the induction and differentiation of defined neuronal phenotypes in mouse ES cells. However, specific isolation or purification of specific target cell types was not achieved in any of these studies. This is a particularly important issue in the use of hES-derived cells for cell-based therapy, since incompletely differentiated hES cells can be potentially tumorigenic upon implantation.
The present invention is directed to satisfying the need for a source of motor neurons from embryonic stem cells.